Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 μg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC–MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 μM) and MCF7 (3.2 μM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.


Introduction
Camptothecin (CPT) is a cytotoxic alkaloid that was firstly isolated from Camptotheca acuminata and Nothapodytes foetida [1], that has been considered as the third commercially prescribed anticancer drug after Taxol and vinblastine [2].The powerful antiproliferative activity of CPT elaborates from its astonishing efficiency to block the eukaryotic topoisomerases activity [3,4], thus preventing the DNA relaxation of tumor cells, unlike to the very trivial effect on the normal cells.In eukaryotic cells, the tyrosine of Topoisomerase I that covalently linked to the 3'-end of the single strand DNA, while topoisomerase II was linked to the 5'-ends of the cleaved double strand DNA, relaxing the DNA supercoiling, allowing to the normal DNA replication and transcription of cells [4][5][6][7].The Topoisomerases I and II causes single and double strand breakages, relaxing the DNA strands, and catalyzes the religation of the cleaved DNA [4].Camptothecin treatment block the activity Topoisomerases consequently stop the DNA replication and cell cycle [8], abolishing the DNA synthesis with ultimate death of cells [9].The biosynthetic pathway of CPT and their ratelimiting biosynthetic enzymes were previously described [10][11][12][13].Currently, Camptotheca acuminata is the major source of CPT, nevertheless, the yield of CPT from the bark and leaves of the plant usually less than ~ 0.4% [14][15][16].So, with the tiny yield of CPT and heavy demand, in addition to limitation of this plant to the Asian geographical niches, an industrial burden has been added to commercially fulfill the required amount of this core compound to the pharmacy [10,11,17].Recently, the metabolic potency of the endophytic fungi of the medicinal plants for production of CPT elevates the prospective for industrial production of CPT, for their fast growth, and feasibility of controlling the fermentation bioprocess [12,13,15,18].The endophyte of Nothapodytes foetida, Entrophospora infrequens, was reported as first CPT producer [19,20], followed by numerous endophytic fungi inhabiting various plants [12,13,18,21,22].Several endophytic fungal isolates namely, Aspergillus terreus an endophyte of Ficus elastica [12], Cestrum parqui [21], Cinnamomum camphora [22], A. flavus, an endophyte of Astragalus fruticosus [13], and Penicillium chrysogenum an endozoic of Cliona sp [18], were affirmed as a promising CPT producing isolates.However, the participation of fungi for the industrial production of CPT usually counteracted by loss of CPT productivity with the subsequent the fungal subculturing and storage [12,13,15,18,[21][22][23][24][25][26].Several approaches have been implemented to restore the CPT biosynthetic machinery of fungi via metabolic engineering by overexpressing their rate limiting genes, via co-cultivation with the microbiome of the host plant, or by addition of different plant extracts [21,25].However, searching for a novel fungal endophyte inhabiting medicinal plants of well-known ethnopharmacological features, with a plausible stability for CPT production is the challenge, in addition to evaluating the antiproliferative and inhibitory effect to of the purified CPT to Topoisomerases.

Collection of plant samples, isolation and identification of their endophytic fungi
The leaves of Catharanthus roseus were obtained from the campus of Zagazig University, in October/2021, and their endophytic fungi were isolated using Potato Dextrose Agar medium (PDA), according to our previous studies [26][27][28].The emerged hyphal tips of fungi were purified, and the fungal isolates were identified based on their morphological features [29][30][31].
The selected CPT producing fungal isolate were molecularly confirmed relied on the sequence of their ITS region [32,33].The genomic DNA was extracted by CTAP [12], used as a template for PCR with the primer set ITS4 5′-GGA AGT AAA AGT CGT AAC AAGG-3′ and ITS5 5′-TCC TCC GCT TAT TGA TAT GC-3′.The PCR reactions contain 10 μl of 2 × PCR master mixture (i-Taq ™ , Cat. # 25027), 1 μl of gDNA, 1 μl of primers (10 pmol/μl), in 20 μl total volume.The PCR was programmed to initial denaturation 94 °C for 2 min, denaturation 94 °C for 30 s, annealing 55 °C for 10 s, extension at 72 °C for 30 s for 35 cycles, and final extension at 72 °C for 2 min.The amplicons were analyzed by agarose gel, sequenced, and the obtained sequences were non-redundantly BLAST searched, aligned by Clustal W [34] and the phylogenetic was analyzed with neighbor-joining method of 50 bootstrap replication [35].

Screening, chromatographic analyses of CPT production by the recovered fungi
The CPT productivity by the endophytic fungi was determined by growing on potato dextrose broth (Cat.#DF0549-17-9) [13,18,23].After incubation of the culture for 10 days at 30 °C under static conditions, the cultures were filtered, extracted with methylene chloride, concentrated with the rotary evaporator till oily residues.The crude fungal extracts were fractionated by 1 mm TLC (Silica gel 60 F 254 , Merck KGaA, Germany) with dichloromethane and methanol (9:1 v/v).The TLC were illuminated at λ 254 nm, and the spots gave the same color and relative mobility of the authentic CPT (Cat.#7689-03-4) has been measured.The CPT spots intensity was assessed by Image J package, regarding to known the concentration of the authentic one.The CPT sample was extracted from the target silica gel spots [12,13,24,36], and the purity and concentration of the extracted CPT was analyzed by the High Performance Liquid Chromatography (YOUNG In, Korea) of C18 column (Cat.#959963-902).The solvent system was methanol/ water (60:40 v/v) at flow rate 1.0 ml/min for 20 min, and the absorbance of the sample was measured at wavelength λ 360 nm.The purity and concentration of the sample was assessed from the retention time and peak area of the authentic CPT [12,36].

UV-Vis, FT-IR, HNMR, and LC-MS/MS
The purified samples were dissolved in methanol, scanned by UV-Vis at λ 200 -λ 500 nm using RIGOL, Ultra-3000 Spectrophotometer, using methanol as a baseline.The authentic CPT was scanned at the same conditions, and the spectroscopic identity of the sample was assigned compared to the authentic one.The FT-IR spectra of the extracted CPT were measured at 400-4000 cm −1 in KBr discs, regarding to the authentic one.As well as, the identity of purified CPT was determined by the 1 HNMR (JEOL, ECA-500II), the shifts and coupling constant (ppm) were determined [12,13,22,24,36].
The nature of the CPT samples were verified by the LC-MS/MS (Thermo Scientific, LCQ Deca mass spectrometer, in a positive mode).The mobile phase was acetonitrile in 0.1% formic acid.The samples were injected to a Hypersil Gold C18 column, with a gradient elution at 2-98% mobile phase at flow rate 0.2 ml/min for 40 min [12,18,24,36].The molecular identity of the target sample was verified from their molecular mass, compared to authentic CPT.Further molecular fragmentation analysis using MS/MS to the selected peaks of the parent molecule, compared to the authentic CPT at 349.1 m/z, were conducted.The molecular identity of the sample was verified from the identity of molecular fragmentation with MS/MS corresponding to the authentic one.

Antiproliferative activity and kinetics of Topoisomerase I, II inhibition by CPT
The anticancer activity of the extracted CPT was assessed towards the breast carcinoma (MCF7, ATCC HTB-22) and Renal cancer cell lines (UO-31, EZT-UO31-1), regarding to the normal oral epithelial cells (OEC) by the MTT assay [37].The cells were cultured on DMEM (Invitrogen) contains 10% FBS, 10 ug/ml of insulin, 50 U/ml of penicillin, and 50 μg/ml of streptomycin.The 96-well plate was seeded with 10 3 / well, incubated at 37 °C for 12 h, then amended with different concentrations of the purified CPT (1.0, 2.0, 4.0, 8.0 and 10 μM) dissolved in 2% DMSO, then incubated for 48 h at the same conditions.DMSO at 2% was used as negative control.The MTT reagent was added, and the purple color was measured at λ 570 nm.The IC 50 value was represented by the CPT concentration suppressing the cellular growth by ~ 50%, compared to the control.
The topoisomerase I activity was measured by converting the supercoiled circular DNA to relaxed one [5], the relaxed DNA reduce the fluorescent intensity of H19 dye, according to the manufacturer's instructions (Cat.#.HRA020K), than the supercoiled one.The assay of Topo I contain HT buffer, 10 × supercoiled plasmid DNA, Dye H19 and 10 × H19 dilution buffer, incubated for 1 h, at different CPT concentrations.One unit of Topo I activity, refers to the amount of enzyme required for relaxing of 1 μM supercoiled DNA at 37 °C per min, and the florescence emission intensity was measured at λ 535 nm at λ 485 nm excitation [6].

Wound healing of the tumor cells in response to the extracted CPT
The wound healing potency of UO-31 cells responsive to CPT was assessed [38,39].The cells were seeded at 5 × 10 6 cells/40 mm well, then allows to grow as a confluent monolayer (about 60 k/cm 2 ), then a scratch was made, the plate was rinsed with PBS, then treated with the purified CPT at the IC 25 values, with DMSO as vehicle.The gap closure due to the migration of the cells was monitored by phase-contrast microscope.The wound healing percentage was assessed relied on the gap area of the treated cells, normalized to the control cells.

Apoptosis and cell cycle analyses of tumor cells in response the extracted CPT
The apoptosis of the UO-31 cells in response to purified CPT was measured with Annexin V-FITC Apoptosis according to the manufacturer's instructions (Cat #: K101-25), that relied on the externalization of phosphatidylserine (PS) of the inner face of plasma membrane that can be easily detected by fluorescent stain Annexin V flow cytometry analysis [40].The UO-31 cells were seeded to 12-well plate culture (2 × 10 6 cells/well), amended with the extracted CPT at the IC 25 values, for 48 h incubation.The cells were washed with phosphate buffered saline, annexin-binding buffer, followed by Annexin V-FITC and PI, according to manufacturer's instructions.The assay was incubated in dark for 15 min, and the Annexin V-FITC binding was detected by flow cytometry (Ex, λ 488 nm; Em, λ 530 nm).
The cell cycle of UO-31 cells was analyzed by Propidium Iodide (PI) Flow Cytometry Kit (Cat #. ab139418).The cells of UO-31 were seeded in 12-well microtiter plate, incubated for 12 h at 37 °C, amended with the IC 25 value of extracted CPT, incubated for 48 h.The cells were collected and fixed in 1 ml of ice-cold 70% ethanol for 2 h at 4 °C, rehydrated with PBS, stained with 500 μl of PI with RNase, for 30 min in dark.The DNA contents of the cells was measured by the flow cytometry at Ex λ 493 nm and Em λ 636 nm, and the ratio of the G0-G1, S and G2-M cells were calculated.

Bioprocessing of the CPT yield by selected fungal isolates with Plackett-Burman design
The physicochemical parameters of the potent isolates were exploited to optimized their yield of CPT with by the Plackett-Burman design [13,18,26,[41][42][43][44].Nineteen variables; malt extract, yeast extract, glucose, sucrose, salicylic acid, asparagine, glutamine, cysteine, tryptophan, glycine, phenylalanine, peptone, pH, incubation time, sodium acetate, citric acid, CaCl 2, NaCl, methyljasmonate were bioprocessed by the Plackett-Burman to increase the CPT yield by the tested fungus.The tested parameters were represented by high (+ 1) and low (-1) levels.The design depends on the first order reaction: Y is the yield of CPT, Xi is the independent variable, βi is the linear coefficient, and β0 is the intercept.The runs were conducted in biological triplicates and the response was expressed by the means of CPT yield.

Metabolic biosynthetic stability of the productivity of CPT by the potent fungal isolates
The biosynthetic stability of CPT by the fungal isolate with the storage and subculturing was assessed [12,18,22,45,46].The axenic CPT-producing fungal culture was successively sub-cultured for 9 generations with a plug centrally inoculated on PDA plate incubated at 30 °C for 8 days lifespan.The CPT productivity was determined by growing the fungus on the optimized media, incubated under standard conditions, and then the CPT was extracted, quantified by TLC.
The axenic 1st culture of the CPT producing isolate was stored as a slope PDA culture at 4 °C, tested for their CPT productivity by growing on PDA media, along 10 months, then the CPT was extracted, quantified as determined above [21,22].

Restoring of the productivity of CPT of Alternaria brassicicola upon addition of organic extracts and indigenous microbiome of C. roseus
In an endeavor to restore the metabolic biosynthetic potency of CPT by Alternaria brassicicola, different organic extracts of C. roseus (methylene chloride, methanol, ethylacetate, petroleum ether, and isopropyl alcohol) were amended to the CPT production medium.The fresh leaves of C. roseus (10 g) were pulverized in the solvents (100 ml) for 12 h, and the extracts were filtered, centrifuged at 5000 rpm, and concentrated to 20 ml.The plant extracts were incorporated to the 3rd day old pre-fungal cultures at concentrations 1, 5 and 10 ml per 50 medium, and the cultures were continued for incubation for 15 days, then the CPT was determined.
The influence of indigenous microbiome of C. roseus leaves on reinstating the productivity of CPT by A. brassicicola was assessed.The leaves of C. roseus were sectioned, surface sterilized and amended into 3 days old culture of A. brassicicola grown on PDB medium, and the cultures were continued for 15 days incubation, then the CPT was assessed by HPLC.The surface sterilized leaves of C. roseus were inoculated into blank PDB media at the same concentrations, and used as control, regarding to the Alternaria brassicicola culture without plant parts.

Statistical analysis
The experiments were conducted in triplicates and the yield of CPT was expressed by the means ± SD.The significance was determined by one-way ANOVA, with Fisher's Least Significant Difference of post hoc test.

Isolation, identification and screening for CPT production from the endophytic fungi of Catharanthus roseus
Fifteen fungal isolates were obtained from C. roseus on the PDA media, and identified according to their morphological features into five genera; Aspergillus, Cladosporium, Alternaria, Trichoderma and Chaetomium.The recovered isolates from the leaves (5 isolates) and twigs (ten isolates), were grown on liquid PDB medium at standard conditions, after incubation, CPT was extracted and quantifie.From the results (Table 1), the maximum CPT yield was reported for Alternaria brassicicola (96.5 μg/L), A. solani (90.5 μg/L), A. alternata sp (67.6 μg/L) and Trichoderma atroviridae (38.5 μg/L).Obviously, the endophytic fugal isolates from the leaves of C. roseus had no the ability to produce CPT, while, the CPT producing isolates were mainly endophytes of the plant twigs, suggesting the presence of some chemical signals derived from the plants or communication of the plant endogenous microbiome on the twigs rather than leaves.The most potent CPT producing endophytic fungal isolate, has been identified based on its macromorphological and microscopical features (Fig. 1), the fungal plate culture appeared olivegrey, grey-black at maturity with velvety appearance, long chain, branched conidiophores with septate hyphae.The conidia were light and dark green in color, with ellipsoidal or ovoid shapes and with or without several longitudinal or oblique septa.The macroscopical and microscopical  The productivity of the most potent CPT producing fungal isolate "Alternaria brassicicola" was preliminary assessed by TLC, then further verified by the HPLC, compared to authentic CPT.From the HPLC chromatograms (Fig. 1F), the crude ethylacetate extract of A. brassicicola had a sharp peak at retention time 5.4 min, that typically matched with the authentic CPT, ensuring the presence of CPT on the extracts.The concentration of the putative CPT of A. brassicicola from the TLC and HPLC chromatogram was mostly identical, normalizing to known concentration of the authentic one for each approach of chromatography.

Chromatographic, spectroscopic analyses, and LC-MS/MS of the extracted CPT
The identity of the purified CPT from A. brassicicola was resolved from the UV-Vis, FTIR, H NMR, and LC-MS/MS analyses.CPT was extracted, fractionated by TLC, and the CPT-containing silica gel spots with the same mobility rate and color of the authentic one, was scraped off, dissolved in methanol for further studies (Fig. 2A).The purified CPT from A. brassicicola had an obvious maximum absorption peak at λ 360 nm, that was completely distinctive to the absorption of the standard one (Fig. 3B), ensuring the physicochemical proximity with the authentic CPT.As well as, from the FTIR spectra (Fig. 2B), the purified A. brassicicola CPT had a distinct absorption peaks at 3406.6 and 3393.3 cm −1 , that refers to the hydroxyl (OH) and amide group stretches.As well as, CPT had a distinct peak of 2923.5, 1729.8 and 1604.5 cm −1 that was refers to the aliphatic CH, ester groups and aromatic rings stretch.The COO stretching frequency peaks at 1268, and 1029 cm −1 were refers to the aromatic C and H blends.The distinct peaks of CPT was resolved at 3438, 1666, 1113 and 1035 cm −1 that assigned to the stretching of OH, C=O, C=N, C-C(=O)-O and C-O functional groups, respectively (Fig. 3C).From the FTIR spectrum, the purified CPT from A. brassicicola had the same functional groups orientation and stretching patterns of authentic one, ensuring the chemical identity of the purified sample as CPT.The identity of CPT from A. brassicicola resolved from the H NMR displayed the identical signals of authentic CPT, distributed at 1.0 ppm and 8.0 ppm, with three proton signals 1.0-2.5 ppm, that assigned to methyl, acetate and acetylene groups (Fig. 3).
The molecular identity of CPT has been authenticated from the LC-MS/MS, the parent CPT molecule of A. brassicicola had the molecular mass to charge ratio (349.2 m/z), that being identical to the authentic CPT from C. acuminata [62].Moreover, the CPT molecule (349.

Antiproliferative, Topoisomerases inhibition, wound healing activity in response A. brassicicola CPT
The anticancer activity of the extracted CPT of A. brassicicola was evaluated towards MCF7, and UO-31 cells, compared to OEC as normal cells.The purified CPT was amended to the RPMI medium of the tumor cells at different concentration, incubated at standard conditions, then the viability of cell were determined by the MTT assay.Practically, the extracted CPT had a strong activity against the viability and multiplication of the tested cells, in a concentration-dependent manner.As revealed from the calculated IC 50 values (Fig. 3A), the A. brassicicola CPT had a dramatic activity towards the UO-31 (0.75 μM), and MCF-7 (33.27 μM) cells, compared to OEC normal cell lines (23.2 μM).The anticancer activity of the putative CPT of A. brassicicola was relatively higher than those reported for the reference drug, by about two folds, as detected from the IC 50 values.The IC 50 values of Staurosporine towards the UO-31, MCF-7 and OEC was 4.23, 7.8 and 27.3 μM, respectively.So, the extracted A. brassicicola CPT displayed a powerful activity towards MCF7 and UO-31 than the authentic anticancer drug "staurosporine".From the IC 50 values, the activity of A. brassicicola CPT towards UO-31 was ~ twofolds higher than MCF7, ensuring the susceptibility of UO-31 to CPT, that might due to feasibility of entrance to the cytosol and binding with Topoisomerases.From the results the selectivity index of the purified CPT of A. brassicicola toward the cell UO-31 and MCF-7 cells was about 30.8 and 7.1 folds, authenticating the selectivity of purified CPT for targeting the tumor cells than the normal ones.
The potency of the purified A. brassicicola CPT to suppress the activity of DNA Topo 1 and II was determined.The standard reaction assay of Topo I, and II were amended with different CPT concentrations, incubated at standard conditions, and the residual enzymes activity was assessed.From the results (Fig. 3B), the IC 50 value of CPT of A. brassicicola towards topoisomerase I and II was 3.2 nM and 0.26 nM, respectively, i.e. by about 12.4 increments folds to inhibit Topo II than I, ensuring the unique structural activity relationship of this compound to suppress the catalytic efficiency of Topo II.The purified A. brassicicola CPT displayed a significant activity towards Topoisomerase II than I by about 12 folds, that being relatively matched with the results of staurosporine.The inhibitory effect of A. brassicicola CPT and staurosporine was obviously similar for the Topoisomerase II, with a noticeable higher activity of CPT for the Topo II by about 12 folds, that might related to the structural activity relationship of CPT for binding with Topo II than I.
The wound healing activity of UO-31 in response to A. brassicicola CPT was evaluated by measuring gap closure after 48 h, compared to the untreated cells.The UO-31 cells were selected for the wound healing activity assay for their sensitivity to the tested CPT.
Obviously, the gap closure percentage was noticeably inhibited upon addition of A. brassicicola CPT, with the incubation time, compared to the control cells (Fig. 3A).Practically, in presence of CPT, the wound healing of the UO-31 cells was about by 45% and 52%, after 24 and 48 h, respectively, compared to the control cells (96%) (Fig. 3B).Thus, upon addition of A. brassicicola CPT, the regeneration of UO-31 cells were suppressed by more than 50% after 24 h, compared to control cells.The remarkable suppression of wound healing ensures the interference of CPT with the cellular machineries of regeneration of the tumor cells UO-31.Interestingly, the purified CPT of A. brassicicola had a noticeable dual activity by binding with topoisomerases I and II, and preventing the matrix formation for the cells motility, that being a therapeutically affordable feature.

Apoptosis and cell cycle analysis of UO-31 in response to CPT of A. brassicicola
The apoptosis of UO-31 in response to CPT of A. brassicicola was assessed based on the externalization of membrane phosphatidylserine by the Annexin V-PI assay, forming Annexin V-PS complex that can be analyzed.From the flow cytometry results (Fig. 4), a significant shift of the normal cells to apoptotic phase was observed in response to the CPT of A. brassicicola, compared to control (untreated) cells.Upon treatment with A. brassicicola CPT, the percentage of the UO-31 cells in early apoptosis, late apoptosis, and necrosis were ~ 26.61, 14.2 and 3.36%, respectively.Unlike to the significant effect of A. brassicicola CPT for induction of the cellular apoptosis, the percentage of early apoptosis, late apoptosis, and necrosis of the untreated cells were 0.58, 0.11 and 1.47%, respectively.So, upon addition of A. brassicicola CPT, the total apoptosis of UO-31 cells was increased by about 20.5 folds, compared to the untreated cells.
The cell cycle of UO-31 was analyzed in response to addition of CPT of A. brassicicola, by propidium iodide assay.The cells were amended with the IC 25 values (0.35 μM) of CPT, incubated, collected, and the percentage of G0-G1, S and G2-M cells were determined.From the cell cycle analysis (Fig. 4), the growth of UO-31 cells was maximally arrested at S-phase by about 47.3%, compared to the without treatment cells (29.7%).However, there is no significant effect by the CT of A. brassicicola has been observed on UO-31 cell at the G0-G1 and G2-M phases.Overall, CPT of A. brassicicola had a noticeable inhibitory effect to the cells at the S-phase, as revealed from the maximum growth arrest, compared to the other cell cycle phases.

Bioprocessing of CPT production by A. brassicicola
The CPT yield by A. brassicicola was augmented via optimization, since the chemical components of the media and their interactions were essential in manipulating the biosynthesis of bioactive secondary metabolites [12,13,18,22,26,43].The requirements for maximum CPT production by A. brassicicola were optimized by Plackett-Burman design.Nineteen parameters including different carbon, nitrogen, growth elicitors, and inhibitors, in addition to the physical factors were tested for CPT production by A. brassicicola, at their lower and higher values (Table 2).The matrix of the Plackett-Burman design for the predicted and actual yield of CPT by A. brassicicola, and their residuals were summarized in Table 3.The yields of CPT by A. brassicicola were strikingly fluctuated from 22.03 to 322.7 μg/L, ensures the significance of the tested variables on CPT productivity, in addition to the proficiency of the design of Plackett-Burman.The main effects of the response surface methodology namely Pareto chart, normal probability, plots of the residuals and the three dimensional surface plots of the variables interactions were shown in Fig. 5. From the Plackett-Burman design, seven independent variables have a significant impact on controlling the CPT biosynthesis by A. brassicicola namely, D-Glucose, L-Asparagine, 32.78 21.9 4 242.44 20.5 205.26 97.5 14 218.61 0.06 17 89.17

Productivity of CPT by A. brassicicola with the subculturing and storage
The metabolic biosynthetic stability of A. brassicicola for CPT with the subculturing and storage was assessed.The 1st isolate of A. brassicicola was conserved as slope PDA cultures for 10 days at 30 °C, followed successive subculturing to the 9th generation, the yield f CPT by the culture was assessed.Practically, a noticeable loss to the CPT productivity by A. brassicicola has been observed with the fungal successive subculturing (Fig. 6).The yield of CPT by the 1st culture of A. brassicicola (316.5 μg/L) was reduced by ~ 2.6 folds by the 6th generation (120.3 μg/L).By the 9th subcultures of A. brassicicola, the yield of CPT was reduced by 4.1 folds (77.1 μg/L), compared to the 1st culture (316.5 μg/L).So, loss of the yield of CPT with successive subculturing of A. brassicicola has been noticeably recorded.
As well as, the influence of storage time of A. brassicicola as slant culture on PDA at 4 °C has been assessed till 12 months.Remarkably, the initial productivity of CPT by A. brassicicola was noticed to be suppressed by about 50%, by the 6th month of storage at 4 °C.The productivity of CPT by the 1st A. brassicicola (316.5 μg/L) culture, was reduced to 93.5 μg/L by the 10th month of storage, i.e. by about ~ 3.4 reduction folds.

Effect of organic solvent extracts and indigenous microbiome of C. roseus on restoring the productivity of CPT by A. brassicicola
Reduction of CPT productivity by fungi with the subculturing and storage is the major metabolic change that limits their further industrial applications [13,22,46,49].The attenuation of CPT productivity by fungi with the fungal subculturing and storage could be associated to some signals from the plant or from their indigenous microbiome.The 6th A. brassicicola culture was supplemented with different extracts of C. roseus, at standard conditions, then the CPT was extracted, and quantified.From the results (Fig. 6C), the extracts of C.

Table 4 Results of ANOVA analysis for the selected factorial model for CPT production by Alternaria brassicicola
The Model F-value of 20.90 implies the model is significant.There is only a 0.01% chance that a "Model F-Value" this large could occur due to noise  roseus "methanol, dichloromethane, ethylacetate, petroleum ether, isopropyl alcohol" had no obvious impact on restoring the CPT productivity by A. brassicicola.The productivity of CPT by the 6th A. brassicicola culture was assessed in response to addition of different plant parts.Interestingly, the yield of CPT by A. brassicicola was restored and strongly enhanced upon addition of the surface sterilized parts of C. roseus leaves.Plant parts without A. brassicicola were used as controls.The productivity of CPT by the 6th A. brassicicola was maximally increased by addition of 1% of C. roseus leaves (310.1 μg/L), i.e. by about 2.4 folds.Thus, upon using the C. roseus microbiome, the productivity of A. brassicicola CPT was restored to be similar with the first fungal culture, might be due to the possibility of the intimate growth with A. brassicicola.

Discussion
CPT has been recognized as one of the most prescribed drugs for treatment of the solid tumors, due to their unique ability to bind with Topo I, thus, stabilizing the DNA-supercoiling, and preventing the relaxation of DNA, leading to ultimate cell death [7,8].The producing potency of CPT by fungi raise the hope for opening a new avenue for industrial CPT production, for their rapid growth, accessibility for mass biomass production, and feasibility of their metabolic engineering, however, the participation of fungi for commercial CPT production was limited by the loss of CPT yield with storage [15,18,22,24,50].So, searching for a novel fungal isolate with higher productivity, affordable biosynthetic CPT stability was the objective.The medicinal plants were considered as a wild repertoire for fungal endophytes with astonishing metabolites of diverse biological activity.Catharanthus roseus, one of the crucial worldwide plants, possessing a wide-range of phytochemicals with diverse antioxidant, antimicrobial and anticancer properties, especially vinblastine and vincristine [51,52].Among the recovered endophytic fungi inhabiting the twigs of C. roseus, A. brassicicola EFBL-NV1 OR131587.1 was most potent CTP producing isolate (~ 97.9 μg/L).Consistently, several isolates of A. terreus, endophytes of F. elastica, C. parqui, A. fruticosus and C. camphora were reported as the potent CPT producers, ensuring the presence of the distinct CPT biosynthetic system, regardless to the diverse plant hosts [12,13,18,21,22,46,53].Interestingly, the potent CPT producing endophytic isolates from the twigs of C. roseus were belonging to three species of Alternaria; A. brassicicola, A. solani, and A. alternata, suggesting the biological role of the plant parts, fungal-host plant interactions, physiological and biochemical identities of the plant microbiome that modulates the expression of the biosynthetic machinery of CPT.Remarkably, the incidence of CPT producing isolates among the various medicinal plants, affirms the reciprocal mechanism of the fungal-plant interaction.Practically, three isolates of Alternaria species were recovered from the twigs of C. roseus, with relatively different CPT yields, assuming the biological identity dependence of the endophyte in correlation with the plant host, interactions of fungal-microbiome, that modulates the expression of the CPT encoding genes by the different species of Alternaria [12, 18-22, 25, 46, 54].Consistently, A. alstroemeriae and A. burnsii, endophytes of Nothapodytes nimmoniana were reported to produce CPT by ~ 403-426 µg/g DW [24].
The chemical identity of the purified CPT of A. brassicicola was resolved by the UV-Vis, FTIR, HNMR and LC-MS/MS, regarding to the authentic one.The molecular mass of the purified CPT was 349 m/z, in addition to the same molecular fragmentation paradigm, as shown from the MS/MS, that was identical to the C. accuminata CPT [12,25,36,55,56].The mass and fragmentation pattern of CPT of A. brassicicola was coincident to that of A. alstroemeriae and A. burnsii, endophytes of Nothapodytes nimmoniana [1,57,58], and A. terreus [12,13,22].Thus, from the FTIR, H NMR, LC-MS/MS analyses, the A. brassicicola sample has been chemically authenticated as CPT.
The purified CPT of A. brassicicola had a significant activity towards the UO-31 (IC 50 0.75 μM), MCF-7 (IC 50 33.27μM) cell lines, compared to the OEC normal cell lines (IC 50 23.2μM), with selectivity index to the UO-31 cells ~ 30.8 folds.The IC 50 value of A. brassicicola CPT towards Topo I and II was 3.2 nM and 0.26 nM, respectively, i.e. by about 12 folds selectivity to Topo II than I.The anticancer activity of the extracted A. brassicicola CPT was coincident with CPT from various endophytic fungi towards the tested cell lines [12,13,18,22].The higher affinity of A. brassicicola CPT for binding with topoisomerase II and I could be an affordable therapeutic criterion, since Topoisomerase II cleavage both DNA strands, since the down-regulation of Topo I has been recognized as an adaptive mechanism of tumor cells to resist the CPT effect [7,8,59].The unique affinity of A. brassicicola CPT to inhibits Topo II than Topo I, could be due to their specific structural activity relationships of stereo-structural conformation of the current CPT, so, further molecular modeling are ongoing to explore their higher affinity to Topo II than I. Consistently, evodiamine, a natural product from C. acuminata has a dual catalytic Topo I/II inhibitor [60].Therefore, targeting both Topo I and II simultaneously lower the potential for the development of tumor resistance [61].Topo II is an effective target for wide spectrum of anticancer drugs "etoposide, doxorubicin, daunorubicin, mitoxantrone [8,[61][62][63].Both Topo I and II are essential in the progression of cell cycle, so blocking the activity of both enzymes simultaneously lead to synergistic anticancer effects.
The wound healing activity of UO-31 in response to A. brassicicola CPT was determined by measuring the gap closure at 48 h compared to the untreated cells.Thus, upon addition of A. brassicicola CPT, the regeneration of UO-31 cells were suppressed by > 50% after 24 h, compared to the untreated cells.The remarkable wound healing suppression ensures the interference of CPT with the cellular machineries of regeneration of the cells.Interestingly, the purified CPT of A. brassicicola had a noticeable dual activity by binding with topoisomerases I and II, and preventing the matrix formation for the cells motility, that being a therapeutically affordable feature, since cell migration has been mainly connected with numerous pathological conditions "tumor invasion, angiogenesis, and metastasis" [22,38,64,65].A significant shift of the cells to apoptotic phase was observed in response to CPT of A. brassicicola.The total apoptosis of UO-31 cells was increased by ~ 20.5 folds with A. brassicicola CPT, compared to the untreated cells.The cell cycle of UO-31 was analyzed in response to CPT of A. brassicicola at the IC 25 values.The growth of UO-31 cells was maximally arrested at S-phase by about 47.3%, compared to the control cells.The growth of UO-31 cells was maximally arrested at S-phase, compared to the control "untreated" cells, as consistently with previous studies [66][67][68].Since cancer cells spend a greater proportion of time in S-phase compared to noncancerous cells, the probability of CPT binding to cancer cells is greater [24,69].
The metabolic biosynthetic stability for CPT production by A. brassicicola with the subculturing and storage was evaluated.The yield of CPT by A. brassicicola was attenuated by ~ 2.6 folds by the 6th generation.The loss of the CPT yield by fungi with the storage is one of the major challenges that halt their implantation to be an industrial avenue for CPT production [10,11,15,24,25,50,70].The productivity of CPT from A. brassicicola still relatively more stable than the CPT yield of F. solani, an endophyte of C. acuminata [53], T. atroviride, an endophyte of C. acuminata [15], A. burnsii, an endophyte of N. nimmoniana [24,69].The obvious relative stability of CPT productivity of A. brassicicola could be attributed to the biological and physiological identity of the host plant C. roseus, the plant-A.brassicicola interactions, and microbiome-A.brassicicola interactions, that positively triggers the CPT biosynthetic gene cluster of A. brassicicola [24,69].The productivity of CPT by A. brassicicola was suppressed by ~ 50% by the 6th month storage at 4 °C.The extracts of C. roseus had no effect on restoring the biosynthesis of CPT by A. brassicicola, as consistent with our previous studies [12,13,15,18,22], negating the presence of inducing signals from the plant host or dilution of these signals with the extraction processes.Interestingly, the CPT yield of A. brassicicola was strongly restored and enhanced upon adding surface sterilized leaves of C. roseus.So, the overall CPT yield by 6th A. brassicicola was increased by ~ 2.4 folds, upon addition of the C. roseus microbiome.So, the most conceivable hypothesis of triggering the CPT biosynthesis, might be due to the presence of indigenous plant microbiome, with subsequent intimate growth with A. brassicicola [46].Similar reports endorsing the dependence of the biosynthetic machinery of CPT by the endophytic fungus on the fungal-plant interactions, some signals from the plant or from their indigenous microbiome [12,13,15,18,21,24,25,69,70].
In conclusion, A. brassicicola, an endophyte of C. roseus, was the most potent CPT producer.The structure of the purified CPT was resolved from the FT-IR, LC-MS/MS, compared to the authentic one.The purified A. brassicicola CPT had a potential antiproliferative activity, with higher affinity to inhibit the Topo I and II, preventing the wound healing of tumor cells, in addition to the induction of the cellular apoptosis.The biosynthetic potency of A. brassicicola CPT was reduced with the multiple-subculturing and storage, however, this metabolic potency was restored with adding the sterilized leaves of C. roseus, emphasizing the role of plant-fungal interaction, microbiome-fungal interactions on triggering the expression cryptic genes of CPT biosynthesis by A. brassicicola.So, transcriptomic and proteomic analyses are ongoing to explore the biosynthetic machinery of CPT by A. brassicicola, to sustain their biosynthetic stability to be a novel platform for industrial CPT production.

Fig. 1
Fig. 1 Morphological and molecular identification of the most potent CPT-producing endophytic fungal isolate inhabiting Catharanthus roseus.A Morphological view of C. roseus.B Plate culture and conidial ontogeny of Alternaria brassicicola as the potent CPT-producing fungal isolate.C PCR amplicons of the ITS region of A. brassicicola, normalized to the DNA ladder (1 kb Nex-gene Ladder, Puregene, Cat.# PG010-55DI).D Molecular phylogenetic analysis of A. brassicicola ITS sequence by the Maximum Likelihood method with MEGA X software package.The cultures of A. brassicicola was incubated, CPT was extracted and fractionated by the TLC (E).The CPT were purified from putative spots on TLC, based on their mobility and color, compared to the authentic CPT.F, HPLC chromatogram of the extracted CPT from A. brassicicola, compared to the authentic CPT.The sample has the same retention time 5.5 min of authentic one

Fig. 2
Fig. 2 Chromatographic and spectroscopic analysis of the purified CPT of A. brassicicola.A TLC chromatogram showing the putative CPT.B UV-spectra of the purified CPT, compared to the authentic one.C FT-IR spectra of the putative CPT.D HNMR spectra of the putative CPT of A. brassicicola.E, The LC-MS analysis of the putative CPT with molecular mass of 349 m/z.F, MS/MS fragmentations of the parent CPT molecule (349 m/z) 2 m/z) was fragmented by MS/MS applying collision energy of 35 electron Volts (eV), the obtained fragments were of molecular mass 57.1, 133.1, 168.8, 181.1, 220.1, 221.01, 249.1, 275.03, 277.05, 292.13, 305.01 and 319.01 m/z, that being identical to the fragmentation pattern of the authentic CPT.From the 1st MS, at 5.56 min with a molecular ion peak at m/z 349.12 [M + H] + was resolved that being refers to the structural formula C 20 H 16 N 2 O 4 .Thus, from the FTIR, H NMR, LC-MS/ MS, the sample of A. brassicicola has been chemically resolved as CPT.

Fig. 3
Fig. 3 Antiproliferative activity, kinetics of inhibition of Topoisomerase I and II and anti-wound healing activity of the purified A. brassicicola CPT.A The IC50 values of the purified CPT against the MCF7 and UO-31 cell lines, compared to the normal OEC, compared to Staurosporine as positive control (reference drug).B Kinetics of inhibition of Topoisomerase I and II by CPT, compared to Staurosporine as reference drug.C Wound healing of the UO-31 cells in response to A. brassicicola CPT after 24 and 48 h, compared to the control cells.After 24 h of growth of UO-31 cells as homogenous monolayer, a scratch was made and the CPT was added to the well at 0.20 μM.D, The percentage of wound healing of the UO-31 cells in response to A. brassicicola CPT, after 24 and 48 h, compared to zero time as control

Fig. 4
Fig. 4 Cell cycle and apoptosis of UO-31 in response to CPT purified A. brassicicola CPT.The cell cycle of UO-31 cells without CPT (A), and treated with A. brassicicola CPT (B), and the overall cellular growth arrest (C) in response CPT compared to control.The apoptotic analysis of UO-31 cells by Annexin-V-PI without CPT (D), with A. brassicicola CPT (E) and the overall apoptotic ratios (F)

8 LFig. 5
Fig. 5 The main effects of the different variables on CPT production by A. brassicicola with the Plackett-Burman design.A Pareto chart illustrates the order of significance of each variable on CPT production by A. brassicicola.Normal plot, and plot of residuals versus predicted response of CPT by A. brassicicola.Three-dimensional surface plots for interactions of the variables for CPT production.The interaction of for glutamine and asparagine (D), cysteine and glutamine (E), incubation time and pH (F), asparagine and salicylic acid (J), sucrose and glucose (H), and tryptophan and cysteine (H) Values of "Prob > F" less than 0.0500 indicate model terms are significant In this case C, F, J, O, P, S, T are significant model terms Values greater than 0.1000 indicate the model terms are not significant If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model

Fig.
Fig. stability of A. brassicicola for CPT production with the subculturing and storage.The cultures of A. brassicicola was incubated at standard conditions, CPT was extracted and quantified by TLC and HPLC.The yield of CPT of A. brassicicola in response to subculturing (A), and storage for 12 months as slope culture (B).The upper panels are the TLC and lower panels were the yield quantified by Image J. The yield of CPT by the 6 th culture generation of A. brassicicola amended with different extracts (C) and surface sterilized leaves of C. roseus (D)

Table 1
Screening for the fungal endophytes from Catharanthus roseus

Table 2
The coded and actual values for the tested variables

Table 3
Matrix of the Plackett-Burman design for optimization of CPT production from Alternaria brassicicola